CN114181884A

CN114181884A - Method for regulating and controlling plant somatic embryogenesis

Info

Publication number: CN114181884A
Application number: CN202111344539.5A
Authority: CN
Inventors: 陈金慧; 鲁路; 托马斯劳克斯; 施季森
Original assignee: Nanjing Forestry University; Albert Ludwigs Universitaet Freiburg
Current assignee: Nanjing Forestry University; Albert Ludwigs Universitaet Freiburg
Priority date: 2021-11-12
Filing date: 2021-11-12
Publication date: 2022-03-15
Anticipated expiration: 2041-11-12
Also published as: CN114181884B; WO2023082486A1

Abstract

The application discloses a method for regulating and controlling plant somatic embryogenesis, wherein immature zygotic embryos of plants are used as explants, and somatic embryogenesis is induced by controlling the co-expression of stem apex meristem stem cell regulating factors miR394 and WUS. The test results of the application show that WUS has a dose effect and a stage effect for promoting somatic embryogenesis, and miR394 can promote WUS to regulate and control shoot apical meristem development, can improve the induction efficiency of WUS on somatic embryogenesis, and further converts Ler Arabidopsis with low probability of somatic embryogenesis into a plant capable of efficiently performing somatic embryogenesis. The method can effectively regulate and control the plant somatic embryogenesis, and has important significance for rapid breeding of important economic plants.

Description

Method for regulating and controlling plant somatic embryogenesis

Technical Field

The application belongs to the technical field of plant tissue culture, and particularly relates to a method for regulating and controlling plant somatic embryogenesis.

Background

Plant regeneration is the process in which plant somatic cells are reprogrammed under the action of exogenous hormones to develop into an independent plant or organ through cell division and differentiation. The regeneration of higher plant plants mainly comprises Organogenesis (Organogenesis), Tissue repair (Tissue repair) and Somatic embryogenesis (Somatic embryo genesis), and the regeneration plants are obtained by Tissue culture, cuttage, grafting and other modes. Organ de novo development is one of the important modes of plant regeneration, including de novo development of stems and roots, and is the process by which isolated or injured plant tissues are induced to grow adventitious buds or adventitious roots. Unlike plant somatic embryogenesis, plant organogenesis requires only the induction of isolated tissues or organs to reform stems and roots without going through a process similar to plant embryogenesis. Tissue repair is the repair or regrowth of a structure capable of performing a biological function in place of an original organ or tissue after the organ or tissue of a plant is damaged or lost. Somatic embryogenesis refers to the process of dedifferentiation of differentiated somatic cells under certain conditions to obtain meristematic capacity and formation of complete plants through redifferentiation, which is a very strong manifestation of cell totipotency and an effective plant regeneration means.

The plant somatic embryogenesis is a process of establishing a new plant individual shape by a way similar to zygotic embryo development under a specific condition that diploid or haploid somatic cells are not subjected to sexual cell fusion, and the transformation from the somatic cells to complete plants is realized under the reprogramming action of a gene expression mode, wherein the process comprises the steps of transformation of the somatic cells into embryonic cells and redifferentiation of the embryonic cells. In recent years, research on plant somatic embryogenesis has made a series of breakthrough progresses, and not only is the preliminary understanding on the transformation of somatic fates in plant embryogenesis realized, but also a molecular mechanism for regulating and controlling the plant somatic embryogenesis process is explored. Research shows that injury signals, stress signals, hormones, transcription factors and epigenetic pathway control factors form an orderly coordinated control channel to control the process of somatic embryogenesis. Somatic embryogenesis not only provides a basic method for studying embryogenesis, but also is widely applied in the agricultural development.

In plants, the induction of somatic embryos using somatic cells is one of the more studied developmental processes. However, the molecular mechanisms controlling somatic embryogenesis are poorly understood. The ability of somatic cells to acquire embryogenesis during somatic embryogenesis apparently involves reprogramming of gene expression. In addition, embryogenic cell differentiation and morphogenesis of the embryo are also dependent on proper or continuous expression of key genes. The use of high throughput sequencing technology increases the number of genes found to be involved in somatic embryogenesis. The rapid development of nucleic acid sequencing technology has facilitated the study of somatic embryogenesis transcriptomes, with the most common observation being differential expression of transcription factors. During somatic embryogenesis, some transcription factors are involved in regulating somatic embryogenesis by inducing somatic cell dedifferentiation or increasing the production of somatic embryos, such as the overexpression of BABY-BOOM (BBM) and WUSHEL (WUS) is sufficient to induce transformation of somatic cells into embryonic cells, promoting somatic embryogenesis [ Somsich M, Je B I, Simon R, et al CLAVATA-WUSHEL signalling in the shooot molecular [ J ]. Development, 2016, 143 (18): 3238.], [ Zuo J, Niu Q W, Frugis G, et al.the WUSCHEL gene proteins targeted-to-electromagnetic transfer in Arabidopsis [ J ]. Plant Journal, 2010, 30 (3): 349-59.].

Transcriptomics information indicates that genes involved in somatic embryogenesis are divided into stress-related genes, growth regulation-related genes, and transcription factors. The plant genome contains a large number (6-10%) of transcription factor-encoding genes. Some of these transcription factors are common to a variety of plant species. During induction of SOMATIC embryos of different species, differentially expressed transcription factors were found to include ABAINSSITIVE 3(ABI3), AGAMOUS LIKE (AGL), BBM, CUP SHAPED COTYLODON (CUC), FUSCA3(FUS3), LEC, WOUND INDUCED DEDIFFERENTIATION (WIND), LEAFY COTYLEDON LIKE (LIL), SOMATIC EMBRBRYOGENESIS RECEPTORLE KINASE1(SERK1), RWP-RK DOMAIN-CONTAINING4(RKD4)/GROUNDED (GRD), VIPAROUS1(VP1), and WUS, etc. In conifers, homologues of some important transcription factors are also involved in somatic embryogenesis, such as SERK1, LEC1, and WOX2, etc., but it is not clear whether they exhibit a functional expression pattern similar to that of angiosperms [ Chu Z, Chen J, Sun J, et al. de novo assembly and comparative analysis of the transgenic case of cultured in branched wheat straw (tri sodium austivum L.) [ J ]. Bmc Plant Biology, 2017, 17 (1): 244.], [ Riechmann J, Heard J, Martin G, et al. Arabidopsis transcription factors: genome-wide comparative analysis of an atmospheric eukaryotes [ J ] Science, 2000, 290 (5499): 2105-10.].

WUS gene as the core regulatory factor of stem cells has central regulatory role in the development of shoot apical meristem and floral meristem [ Knauer S, Holt A L, Rubio-Somoza I, et al. A protodermal miR394signal definitions a region of stem Cell competition in the Arabidopsis shoot scaffold molecule [ J ]. development Cell, 2013, 24 (2): 125-32.]. Early studies have found that WUS loss-of-function mutations lead to premature termination of the development of shoot apical meristem after several leaves are formed in Arabidopsis, and that mutant flowers terminate meristem without formation of pistils. In addition to participating in regulating embryonic development processes, the WUS gene also plays an important role in somatic embryogenesis. Research shows that the WUS gene is closely related to the transformation from somatic cells to embryonic cells or the maintenance of embryonic cell characteristics in the in vitro culture process, and the over-expression of the WUS gene can promote spontaneous somatic embryo generation without the action of plant hormones.

At present, somatic embryogenesis has been considered to be a common phenomenon in the plant kingdom. As model plants, Arabidopsis somatic embryogenesis research can provide a reference system for the research of somatic embryogenesis of non-model plants. Although somatic embryogenesis has great potential in plant propagation and commercial applications, there are very few plant species that can spontaneously form somatic embryos due to their different somatic embryogenesis capabilities, and many important plants are difficult to somatic embryogenesis or induce low efficiency in somatic embryo formation, including coconut palm, barley of different genotypes, and most conifers, among others. The genotype, explant, and various growth regulators, etc., all affect the efficiency of somatic embryogenesis. The mechanisms underlying these findings remain elusive, and therefore most plants are experientially experimented with appropriate systems to improve the efficiency of somatic embryogenesis.

With the continuous improvement of a somatic embryo induction system and the improvement of induction efficiency, a plant regeneration mode obtained through somatic embryogenesis is often applied to aspects of genetic engineering breeding, seedling propagation, germplasm resource preservation, artificial seed preparation and the like. Moreover, with the rapid development of genome editing technology, most species urgently need an efficient genetic transformation system to obtain gene editing progeny plants. Although organogenesis has been widely used for in vitro propagation and genetic transformation, the characteristic that somatic embryogenesis can be derived from a single cell is considered to be an ideal way of genetic transformation. In addition, somatic embryogenesis is also recognized as a tool for the propagation of economical plants with superior genetic traits. Meanwhile, somatic embryogenesis is also considered to be the least costly method for rapidly producing high value seedlings with the characteristics required for plantation and forestry. In addition, somatic embryogenesis studies are of reference for the development of zygotic embryos. Early in zygotic embryo development, the embryo body is small and surrounded by surrounding maternal cells and is therefore inaccessible. And the zygotic embryo and the surrounding cells thereof have interaction, which brings much difficulty to the research of the development of the zygotic embryo, and leads to relatively slow research progress. Somatic embryo development is completed by culturing under an in vitro condition and is highly similar to the morphogenesis of zygotic embryos, so that the whole process of zygotic embryo development can be reproduced by utilizing the somatic embryo development process, and the difficulty in researching zygotic embryo development is overcome to a certain extent. In addition, the somatic cell generation process can be tracked in real time, conditions can be well controlled, material taking is not limited, and research is facilitated. Therefore, the study of the mechanism of somatic embryogenesis is of practical significance for revealing the developmental mechanism of zygotic embryos.

Disclosure of Invention

Aiming at the defects in the prior art, the technical problem to be solved by the application is to provide a method for regulating and controlling the somatic embryogenesis of a plant, and the regulation and control of the somatic embryogenesis can be effectively realized.

In order to solve the technical problem, the technical scheme adopted by the application is as follows:

a method for regulating and controlling plant somatic embryogenesis takes a plant immature zygotic embryo as an explant, and induces the somatic embryogenesis by regulating and controlling the co-expression level of miR394 (precursor gene ID: AT1G76135) and WUS (gene ID: AT2G 17950).

According to the method for regulating and controlling the embryogenesis of the plant somatic cell, miR394 promotes the formation of the somatic embryo through a WUS-GR protein induced by a synergistic concentration gradient DEX.

The method for regulating and controlling the embryogenesis of the plant somatic cell has DEX concentration of not more than 10 mu M.

The method for regulating and controlling the embryogenesis of the plant somatic cell has DEX concentration of 0.1 nM-0.1 MuM.

The method for regulating and controlling the embryogenesis of the plant somatic cell has the DEX concentration of 1 nM.

The method for regulating and controlling the somatic embryogenesis of the plants promotes the formation of the somatic embryos by regulating and controlling the co-expression quantity of miR394 and WUS at the early stage of the somatic embryogenesis.

Application of miR394 in regulation and control of plant somatic embryogenesis.

Application of miR394 to regulation and control of plant somatic embryogenesis in cooperation with expression of WUS.

Has the advantages that: compared with the prior art, the test result of the application shows that:

(1) the WUS protein has a dose effect on the regulation of somatic embryogenesis, and a proper amount of the WUS protein is an important influence factor for regulating the somatic embryogenesis. A proper amount of WUS protein promotes the formation of somatic embryos, and Lers which are difficult to obtain somatic embryos are initiated to perform somatic embryogenesis; excess WUS protein induced callus formation.

(3) The WUS protein has a phasic effect on the regulation of somatic embryogenesis. Test results show that the WUS can regulate and control the early stage of somatic embryogenesis and has a promoting effect on the efficiency of somatic embryogenesis; in contrast, using WUS to regulate late somatic embryogenesis, Ler cannot be efficiently initiated for somatic embryogenesis.

(4) miR394 can cooperate with WUS to further promote the formation of somatic embryos, the function deficiency of miR394 inhibits the promoting effect of WUS on the formation of Ler somatic embryos, and the efficiency of Col Arabidopsis somatic embryogenesis is reduced, so that the important regulation and control effect of miR394 on somatic embryogenesis is proved.

In conclusion, the test results of the application show that WUS has a dose effect and a stage effect on the promotion of somatic embryogenesis, and miR394 not only can promote WUS to regulate the development of shoot apical meristems, but also can improve the induction efficiency of WUS on somatic embryogenesis, so that Ler Arabidopsis with low possibility of somatic embryogenesis is converted into a plant capable of efficiently carrying out somatic embryogenesis. The method can effectively regulate and control the plant somatic embryogenesis, and has important significance for rapid breeding of important economic plants.

Drawings

FIG. 1 is a graph of the results of a concentration gradient DEX regulation of WUS-GR inducible somatic embryogenesis first stage phenotype; in the figure, the Ler control groups (a-D) were treated with Mock, 1nM, 10nM and 10 μ M DEX, p 35S: WUS-GR (E-H), p 35S: MIR394B/p 35S: WUS-GR (I-L) and pRPS 5A: MIM394/p 35S: WUS-GR (M-P) somatic embryogenesis first stage induction results; arrows indicate sites of apparent cell proliferation; scale bar: 0.05 cm;

FIG. 2 is a graph of the results of a second phase phenotype of WUS-GR protein induced somatic embryogenesis by concentration gradient DEX regulation; in the figure, the Ler control groups (a-D) were treated with Mock, 1nM, 10nM and 10 μ M DEX, p 35S: WUS-GR (E-H), p 35S: MIR394B/p 35S: WUS-GR (I-L) and pRPS 5A: MIM394/p 35S: WUS-GR (M-P) somatic embryogenesis second stage induction results. Arrows indicate immature embryos; scale bar: 0.1 cm;

FIG. 3 is a graph of the results of statistics for the callus induction stage miR394 in conjunction with WUS regulation of immature somatic embryogenesis; "+" indicates p < 0.05, "+" indicates p < 0.01, "+" indicates p < 0.001;

FIG. 4 is a graph of the results of miR394 synergy concentration gradient DEX-induced concentration gradient WUS-GR protein regulation of somatic embryogenesis; in the figure, Ler control group (a-D), p 35S: WUS-GR (E-H), p 35S: MIR394B/p 35S: WUS-GR (I-L) and pRPS 5A: MIM394/p 35S: somatic embryogenesis results obtained by induction of WUS-GR (M-P) explants; arrows indicate somatic embryos; scale bar: 0.1 cm;

FIG. 5 is a graph of the statistics of the number of somatic embryogenesis regulated by miR 394-promoted concentration gradient DEX-induced concentration gradient WUS-GR protein; "+" indicates p < 0.05, "+" indicates p < 0.01, "+" indicates p < 0.001;

fig. 6 is CLV 3: results plot of GUS expression pattern in somatic embryogenesis material; in the figure, after different concentrations of DEX treated WUS-GR, pCLV 3: GUS in the Ler control group (A-D), p 35S: WUS-GR (E-H), p 35S: MIR394B/p 35S: WUS-GR (I-L) and pRPS 5A: MIM394/p 35S: expression pattern in somatic embryogenesis material induced by WUS-GR (M-P) explants; blue represents CLV 3: GUS staining area; scale bar: 0.1 cm;

FIG. 7 is a graph of somatic embryo results obtained from DEX-induced WUS-GR action on the first and second stages of somatic embryogenesis; in the figure, Ler control group (a-D), p 35S: WUS-GR (E-H), p 35S: MIR394B/p 35S: WUS-GR (I-L) and pRPS 5A: MIM394/p 35S: somatic embryogenesis results obtained by induction of WUS-GR (M-P) explants in the first and second stages of DEX action and somatic embryogenesis; scale bar: 0.1 cm;

FIG. 8 is a graph of statistical somatic embryo efficiency obtained during the first and second stages of somatic embryogenesis induced by DEX WUS-GR; "+" indicates p < 0.05, "+" indicates p < 0.01, "+" indicates p < 0.001;

FIG. 9 is a graph of statistics of the number of somatic embryos obtained after 1nM DEX induced the WUS-GR protein to act at different stages of somatic embryogenesis; "x" indicates p < 0.01, "x" indicates p < 0.001;

FIG. 10 is a graph showing the reduction of somatic embryo induction efficiency due to the deletion of endogenous MIR394B function; in the figure, (A) Col wild type and (B) mir394B-1 mutant explants induced a somatic embryogenesis phenotype; (C) col wild type and mir394b-1 somatic embryo formation number statistics. Data analysis one-way ANOVA test was used, "-" indicates p < 0.05.

Detailed Description

The present application is further described with reference to specific examples. The procedures and conditions not described in detail in the following examples can be performed by the conventional procedures or kit instructions in the art.

Example 1

The plant materials and culture conditions used in this example were:

experimental materials in the Ler arabidopsis background:

(1)p35S：WUS-GR，pCLV3：GUS；

(2)p35S：MIR394B，p35S：WUS-GR，pCLV3：GUS；

(3)pRPS5A：MIM394，p35S：WUS-GR，pCLV3：GUS.

the transgenic lines were provided by professor Thomas Laux, university of fleabag, germany.

Experimental materials in the background of Col arabidopsis thaliana:

(1) mir394b-1 mutant plants;

the strain and the wild type arabidopsis seeds are planted in nutrient soil, are placed in a dark environment at 4 ℃ for vernalization for 3-4 days, and are then placed in a light incubator with a light environment of 16h light/8 h dark for germination, wherein the culture environment temperature is 22-23 ℃. Watering for 2-3 times every week to maintain normal growth of Arabidopsis.

2. Experimental methods used in the examples

(1) And (3) positive plant identification:

after the plant grows up, taking the leaves to extract DNA, and detecting the positive plant by PCR. The results show that the transgenic plants used in the experiment are separated in asexual shape, namely all the transgenic plants are positive homozygous transgenic plants.

Identifying primers of positive transgenic plants in Ler background:

TABLE 1 genotyping detection primers

mir394b-1 mutant detection primers and method:

TABLE 2 mir394b-1 mutant identification primer

After a PCR product (155bp) with a target size is obtained through PCR reaction, Msel enzyme digestion is carried out for 4h, and the size of a mutant band is 127bp +28bp as shown by gel electrophoresis; if the size of the band is not changed after the enzyme digestion, the mutant is a non-mir 394b-1 mutant.

(2) Somatic embryogenesis induction Using immature embryos as starting Material

Sterilizing pods in the embryo period of the curvularia glauca with arabidopsis thaliana, and specifically: placing the pod of Arabidopsis thaliana in a triangular flask, treating with 1% sodium hypochlorite solution for 12-15min, and pouring off the solution; washing the pod 3-5 times with sterile water for later use.

Taking out the walking stick embryos under a dissecting mirror, placing the walking stick embryos on a culture medium for somatic embryogenesis induction, and performing Col background arabidopsis thaliana somatic embryo induction steps and culture conditions: b5+4.5 mu M2.4-D solid medium, 16h of light/8 h of dark environment, 22 ℃ and 10 days of culture; b5+9 mu M2.4-D solid medium, culturing for 14 days in a dark environment at the temperature of 22 ℃; b5+ 0. mu.M 2.4-D solid medium, cultured in dark at 22 ℃ for 10 days.

Induction of arabidopsis somatic embryos in Ler background increased DEX treatment: b5+4.5 mu M2.4-D solid medium + Mock or DEX, 16h light/8 h dark environment, 22 ℃ temperature, 10 days of culture; b5+9 mu M2.4-D solid medium + Mock or DEX, culturing for 14 days in a dark environment at the temperature of 22 ℃; b5+ 0. mu.M 2.4-D solid medium + Mock or DEX, in dark at 22 ℃ for 10 days.

In experiments using the DEX/GR inducible system to modulate WUS-GR induced somatic embryogenesis, DEX was used at concentrations of: 0. mu.M, 0.1nM, 1nM, 10nM, 0.1. mu.M, 1. mu.M and 10. mu.M.

(3) Addition of DEX treatment induced WUS-GR at various stages of somatic embryogenesis, the specific stages are shown in Table 3.

TABLE 3 WUS-GR induction by addition of 1nM DEX (or Mock) at different stages of somatic embryogenesis

T1

T2

T3

T4

T5

T6

T7

First stage

+

Second stage

+

The third stage

+

Note: T1-T7 is a treatment mode of adding 1nM DEX at different stages; "+" refers to the addition of a DEX processing stage.

(4) Somatic embryo inducing material GUS staining and microscopic observation

The induced material from each stage was stained with GUS (GUS solution: 0.2% Triton-X-100, 50mM NaPO4(pH 7.2), 2mM Ferro-K, 2mM Ferri-K, 2mM X-Gluc) in a 2mL centrifuge tube, according to the following protocol: (1) plant tissues were collected with 90% acetone on ice; (2) incubating at room temperature for 20-30 min; (3) removing the acetone and rinsing the plant tissue with a dye solution free of X-Gluc; (4) removing the rinsing liquid, and adding a dyeing liquid containing X-Gluc to perform GUS dyeing; (5) vacuumizing the plant tissues soaked with GUS dye solution for 30 min; (6) after vacuum treatment, the plant tissues are placed in a dark environment at 37 ℃ for incubation, and the GUS staining degree is observed at any time, so that process staining is avoided; (7) after dyeing is finished, removing the dye solution, and then adding 70% ethanol solution to stop dyeing; (8) removing 70% ethanol solution, and adding 100% ethanol; (9) removing 100% ethanol, sequentially adding 10%, 20% and 30% … … 100% ethanol solution, and fixing color; (10) taking out the tissue, removing surface alcohol, placing on a glass slide with transparent liquid, performing transparent treatment for 2-4 h, and taking the material to observe and photograph under a stereoscopic microscope.

3. miR394 improves efficiency of inducing somatic embryogenesis by proper WUS protein

The first stage induction of somatic embryogenesis showed that overexpression of WUS alone (p 35S: WUS-GR) induced immature embryos to form callus-like structures in shoot apical meristems and hypocotyl regions at 1nM DEX treatment (FIG. 1F), callus-forming regions expanded with increasing DEX concentration, the entire shoot apical meristem and hypocotyl were covered with callus at 10nM DEX treatment (FIG. 1G), and callus was formed in the cotyledon, shoot apical meristem and hypocotyl regions of immature embryos at 10. mu.M DEX treatment (FIG. 1H). Similarly, in case of co-overexpression of MIR394B and WUS (p 35S: MIR394B/p 35S: WUS-GR), callus formation was observed in hypocotyl and shoot apical meristems of immature embryos by treatment with 1nM DEX, formation of a small amount of callus was observed in cotyledon sites in addition to shoot apical meristem and hypocotyl of immature embryos by treatment with 10nM DEX, and callus coverage was observed in the whole immature embryo except for the cotyledon top in treatment with 10. mu.M DEX. In addition, when MIM394 and WUS were co-overexpressed (pRPSSA: MIM 394/P35S: WUS-GR), the range of immature embryogenic callus was mainly concentrated in the shoot apical meristem and hypocotyl region under DEX treatment of concentration gradient (FIG. 1M-P). However, in the first stage of somatic embryogenesis, cell proliferation was observed around the apical meristem in the wild type Ler control group (FIGS. 1A-D), and no significant phenotypic change occurred at the radicular end, which is significantly different from the structure of transgenic plants forming similar callus at the radicular end.

In the second stage of somatic embryogenesis, the 9. mu.M 2, 4-D induced callus formation results showed that all explants of the test plants, including the Ler wild type, formed callus (FIG. 2). However, under DEX treatment of concentration gradient, callus obtained by induction of overexpression of WUS alone (p 35S: WUS-GR) gradually increased with increasing DEX concentration; WUS alone induced over-expression to form white callus and a small number of immature embryos at 1nM DEX treatment; overexpression of WUS alone induced explants to form an average of 2.57 immature embryos upon 10nM DEX treatment; overexpression of WUS alone induced formation of pale yellow callus at 10. mu.M DEX treatment, and cell proliferation of callus was significantly greater than that induced by WUS protein at 1nM DEX treatment (FIGS. 2F-H). Similarly, in miR394 and WUS co-overexpressed (p 35S: MIR394B/p 35S: WUS-GR) plants, 10. mu.M high concentration of DEX induced formation of pale yellow callus (FIG. 2L), whereas 1nM DEX and 10nM DEX treatment induced explants, in addition to callus formation, on average 9.68 and 5.55 immature embryos (FIG. 2J, FIG. 2K and FIG. 3), respectively, and the number of immature embryos was significantly higher than the number of immature embryos induced when WUS alone was overexpressed (p 35S: WUS-GR) (FIG. 3). However, in the callus induction phase, MIM394 and WUS co-overexpression (pRPSSA: MIM 394/P35S: WUS-GR) plant explants induced only callus formation, no immature embryos, and callus volumes increased gradually with increasing DEX concentrations, under the various concentrations of DEX treatment conditions used (FIG. 2M-P and FIG. 3).

In the third stage of somatic embryogenesis, callus was transferred to auxin-free B5 medium for somatic embryo induction culture. The results show that concentration-gradient DEX treatment of WUS-GR induces somatic embryogenesis with varying efficiencies. Three transgenic plants: WUS alone overexpression (p 35S: WUS-GR) plants, miR394 and WUS co-overexpression (p 35S: MIR394B/p 35S: WUS-GR) plants, MIM394 and WUS co-overexpression (pRPS 5A: MIM394/p 35S: WUS-GR) plants, wherein the number of somatic embryos obtained by exophyte induction is increased firstly and then decreased with the increase of DEX concentration, and the number of somatic embryos obtained by 10nM DEX treatment is the highest, wherein the explant obtained by WUS alone overexpression can induce 3.98 somatic embryos on average (FIG. 4G and FIG. 5); the co-overexpression of miR394 and WUS induces explants with an average of 10.4 somatic embryos, and the number of somatic embryos is remarkably increased compared with that induced by the independent overexpression of WUS (figure 4K and figure 5); in contrast, MIM394 and WUS co-overexpression induced a reduction in the number of embryos compared to WUS overexpression alone (fig. 4O), with explants that obtained an average of 2.19 embryos (fig. 5).

Moreover, co-overexpression of miR394 and WUS induced higher numbers of somatic embryos than those induced by over-expression of WUS alone at each DEX concentration used, lower numbers of somatic embryos induced by co-overexpression of MIM394 and WUS than those induced by over-expression of WUS alone (fig. 5), whereas wild-type Ler explants induced very low numbers of somatic embryos relative to transgenic plants, explants induced up to 0.028 somatic embryos on average, and most explants induced only callus covered by hairy structures (fig. 4A-D and fig. 5).

This example detects pCLV3 by GUS staining: GUS, analyzing the expression level of the WUS protein target gene CLV3 in the third stage of somatic embryogenesis. From CLV 3: GUS expression patterns in plants overexpressing WUS alone (p 35S: WUS-GR), miR394 and WUS co-overexpressing (p 35S: MIR394B/p 35S: WUS-GR) and MIM394 and WUS co-overexpressing (pRPS 5A: MIM394/p 35S: WUS-GR) plants explant induced CLV 3: GUS was expressed in increasing amounts with increasing concentrations of DEX (FIG. 6). Similar to the results of second stage induction of somatic embryogenesis, overexpression of WUS alone induced higher expression of CLV3 than did overexpression of MIM394 and WUS together induced higher expression of CLV 3.

As described above, concentration-gradient DEX treatment of WUS-GR led to a gradual increase in WUS protein induction of calli characterized by CLV3, but the number of embryos obtained by induction increased first and then decreased, MIM394 inhibited WUS-induced somatic embryo formation, and conversely, miR394 promoted WUS-induced somatic embryo formation. These results indicate that WUS protein induction of somatic embryogenesis has a dose effect, that WUS overexpression can indeed promote embryogenic callus formation, but that only a relatively modest concentration of WUS protein can induce somatic embryo formation, and that miR394 can promote a modest WUS regulation of somatic embryo formation.

4. WUS on early regulation of somatic embryogenesis in Arabidopsis

First, in the first two stages of somatic embryogenesis, treatment with a concentration gradient DEX was performed, and no DEX was added in the third stage, as shown in FIGS. 7 and 8, after treatment with 10nM DEX, the number of somatic embryos induced by WUS alone (p 35S: WUS-GR) explants was 4.29 on the mean, and the number of somatic embryos induced by 10. mu.M DEX was significantly reduced compared to the number of somatic embryos induced by low concentration DEX (FIG. 7H), the mean of which was 2.69 (FIG. 8); similarly, under the action of 10nM DEX, the mean number of somatic embryos induced by miR394 and WUS co-overexpression (p 35S: MIR394B/p 35S: WUS-GR) plant explants is 10.21, which is significantly higher than the induction result of WUS alone overexpression plant explants under the same condition (FIG. 8), but under the action of 10 μ M DEX, the number of somatic embryos induced by miR394 and WUS co-overexpression is also significantly reduced, the number of somatic embryos induced by explants is 2.46 on average, and the difference with the induction result of WUS alone overexpression plant explants under the same condition is not significant (FIG. 8). However, at each concentration of DEX treatment used, MIM394 and WUS co-overexpression (p 35S: WUS-GR/p 35S: MIM394) induced predominantly callus, with only a small number of somatic embryos formed; under 1nM DEX treatment, MIM394 and WUS co-overexpression induced the highest mean number of somatic embryos, explants averaged 1.77 somatic embryos (fig. 7J-I and fig. 8), which was lower than the number of somatic embryos induced by WUS overexpression alone at the respective DEX concentrations used, significantly lower than the number of somatic embryos induced by miR394 and WUS co-overexpression (fig. 8).

In addition, the data statistics and analysis result shows that under the treatment mode that DEX only acts on the first and second stages of somatic embryogenesis, the somatic embryo efficiency obtained by induction of three transgenic plant explants is similar to the induction result obtained by the induction of DEX on the whole somatic embryogenesis stage, and the number of the somatic embryos obtained by induction of the two DEX treatment modes has no difference significance (FIG. 8 and FIG. 5). These results indicate that the presence or absence of DEX in the third stage induces WUS-GR regulated somatic embryogenesis with less effect on somatic embryo formation.

WUS-GR protein was induced by the addition of 1nM DEX at different stages of somatic embryogenesis and the efficiency of somatic embryogenesis induced by it was counted. As a result, it was found that when DEX was added only in the first stage or the second stage of somatic embryogenesis, the number of induced somatic embryos was higher than that obtained when DEX was added only in the third stage (fig. 9); when the transgenic plants and wild-type Ler were induced to form callus in the first two stages of somatic embryogenesis without addition, i.e., only 4.5 μ M and 9 μ M of 2, 4-D in B5 medium, and in the third stage in B5 basal medium supplemented with 1nM DEX, the results showed that the number of somatic embryos formed from the transgenic plant explants was low, and on average only 0.06-0.34 somatic embryos were induced per explant, indicating that induction of WUS-GR with DEX alone in the somatic embryogenesis stage did not effectively initiate Ler-formed somatic embryos (FIG. 9).

The treatment mode of adding DEX to induce WUS-GR in the first stage and the second stage leads to the highest somatic embryo efficiency obtained by somatic embryogenesis of transgenic plants (figure 9). Moreover, in the mode of treating different somatic embryogenesis stages with 1nM DEX, both immature embryos co-overexpressed by miR394 and WUS were induced with the highest efficiency of somatic embryos, and both immature embryos co-overexpressed by MIM394 and WUS were induced with the lowest number of somatic embryos. These results further demonstrate the promoting effect of miR394 on WUS regulation of somatic embryogenesis, in contrast to MIM394 inhibiting the promoting effect of WUS on somatic embryogenesis.

In conclusion, WUS overexpression promotes Ler to perform somatic embryogenesis at a key stage, namely an early induction stage of somatic embryogenesis, namely a callus formation stage induced by auxin action, so that the effect of WUS on regulating the stage of somatic embryogenesis is proved. Moreover, miR394 can improve the somatic embryogenesis efficiency of the WUS protein after acting on different stages, and promote the formation of somatic embryos induced by the WUS protein.

5. Endogenous MIR394B regulates somatic embryogenesis efficiency

The test result of regulating somatic embryogenesis by using the concentration gradient DEX induced concentration gradient WUS protein shows that the WUS has a dosage effect on promoting somatic embryogenesis, and miR394 remarkably improves the somatic embryogenesis efficiency induced by a proper amount of WUS. However, under Mock treatment conditions, the WUS-GR fusion protein is theoretically cytoplasmic, rendering the WUS protein unable to exert the regulatory role of transcription factors, therefore, miR394 and WUS co-overexpress plant p 35S: MIR394B/p 35S: only MIR394B was overexpressed and functional in WUS-GR. However, the results show that miR394 overexpression alone did not significantly increase somatic embryo formation (fig. 4I and 5). From this, it is clear that overexpression of miR394 alone cannot initiate somatic embryogenesis of wild-type Ler. However, the efficiency of MIM394 and WUS co-overexpression to induce somatic embryogenesis was lower than that induced by WUS overexpression alone (fig. 5), and this result suggests that MIM394 overexpression acts to inhibit WUS from promoting somatic embryogenesis, in other words, WUS overexpression alone promotes the formation of somatic embryos and the action of endogenous miR394 is correlated.

Columbia (Col) wild type and mir394b-1 mutant Arabidopsis thaliana were used for somatic embryogenesis experiments. Unlike Ler somatic embryogenesis, Col wild type Arabidopsis thaliana can be induced to obtain a certain amount of somatic embryos under the somatic embryogenesis method used in this experiment. The results show that there was no significant difference in the induction status of wild type Col and mir394b-1 after the first and second induction steps of somatic embryogenesis were completed. However, in the third stage of somatic embryogenesis, the number of somatic embryos induced by MIR394b-1 callus is significantly reduced compared with that induced by wild-type Col (FIGS. 10A-C), the number of somatic embryos induced by explants is 1.21 on average, and is significantly lower than that induced by wild-type Col explants by 2.61, so that the expression of endogenous MIR394B plays an important role in regulating and controlling the efficiency of somatic embryogenesis.

Claims

1. A method for regulating and controlling plant somatic embryogenesis is characterized in that immature zygotic embryos of plants are used as explants, and somatic embryogenesis is induced by regulating and controlling the co-expression quantity of miR394 and WUS.

2. The method of modulating somatic embryogenesis in a plant of claim 1, wherein miR394 promotes the formation of somatic embryos via a synergistic concentration gradient DEX-induced WUS-GR protein.

3. The method of modulating somatic embryogenesis in a plant of claim 2, wherein the concentration of DEX is no greater than 10 μ M.

4. The method of modulating somatic embryogenesis in a plant according to claim 2 or 3, wherein the DEX concentration is between 0.1nM and 0.1. mu.M.

5. The method of modulating somatic embryogenesis in a plant according to claim 4, wherein the concentration of DEX is 1 nM.

6. The method for regulating plant somatic embryogenesis of claim 1, wherein somatic embryo formation is promoted by regulating the co-expression level of miR394 and WUS at an early stage of somatic embryogenesis.

Application of miR394 in regulation and control of plant somatic embryogenesis.

Application of miR394 and WUS coordinated expression in regulation of plant somatic embryogenesis.